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. 2012 Sep;135(Pt 9):2750-65.
doi: 10.1093/brain/aws193. Epub 2012 Aug 21.

Late-onset Parkinsonism in NFκB/c-Rel-deficient mice

Cristina Baiguera  1 , Manuela Alghisi, Annalisa Pinna, Arianna Bellucci, Maria Antonietta De Luca, Lucia Frau, Micaela Morelli, Rosaria Ingrassia, Marina Benarese, Vanessa Porrini, Michele Pellitteri, Giuseppe Bertini, Paolo Francesco Fabene, Sandra Sigala, Maria Grazia Spillantini, Hsiou-Chi Liou, Pier Franco Spano, Marina Pizzi
Affiliations

Late-onset Parkinsonism in NFκB/c-Rel-deficient mice

Cristina Baiguera et al. Brain. 2012 Sep.

Abstract

Activation of the nuclear factor κB/c-Rel can increase neuronal resilience to pathological noxae by regulating the expression of pro-survival manganese superoxide dismutase (MnSOD, now known as SOD2) and Bcl-xL genes. We show here that c-Rel-deficient (c-rel(-/-)) mice developed a Parkinson's disease-like neuropathology with ageing. At 18 months of age, c-rel(-/-) mice exhibited a significant loss of dopaminergic neurons in the substantia nigra pars compacta, as assessed by tyrosine hydroxylase-immunoreactivity and Nissl staining. Nigral degeneration was accompanied by a significant loss of dopaminergic terminals and a significant reduction of dopamine and homovanillic acid levels in the striatum. Mice deficient of the c-Rel factor exhibited a marked immunoreactivity for fibrillary α-synuclein in the substantia nigra pars compacta as well as increased expression of divalent metal transporter 1 (DMT1) and iron staining in both the substantia nigra pars compacta and striatum. Aged c-rel(-/-) mouse brain were characterized by increased microglial reactivity in the basal ganglia, but no astrocytic reaction. In addition, c-rel(-/-) mice showed age-dependent deficits in locomotor and total activity and various gait-related deficits during a catwalk analysis that were reminiscent of bradykinesia and muscle rigidity. Both locomotor and gait-related deficits recovered in c-rel(-/-) mice treated with l-3,4-dihydroxyphenylalanine. These data suggest that c-Rel may act as a regulator of the substantia nigra pars compacta resilience to ageing and that aged c-rel(-/-) mice may be a suitable model of Parkinson's disease.

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Figures

Figure 1
Figure 1
Age-dependent loss of dopaminergic neurons in the substantia nigra of c-rel−/− mice. Representative pictures of tyrosine hydroxylase staining in the substantia nigra dopaminergic neurons of wild-type and c-rel−/− mice at 2 months (A and B), 12 months (D and E) and 18 months (G and H). The data from stereology analysis of tyrosine hydroxylase-positive cells are shown in C, F and I. A significant decrease of the number of tyrosine hydroxylase-positive neurons was detected in 18-month-old c-rel−/− mice. The data represent the means ± SEM (n = 8 mice/group, *P < 0.05 versus wild-type mice). Representative pictures of Nissl staining in the substantia nigra of 18-month-old wild-type (L) and c-rel−/− mice (M) confirmed the loss of neurons. Data from stereology analysis are reported in N. Scale bar in B = 350 µm and applies to A–M. TH = tyrosine hydroxylase.
Figure 2
Figure 2
Tyrosine hydroxylase staining in the ventral tegmental area, ChAT staining in the medial septal area and nucleus basalis magnocellularis, NeuN staining in the striatum, of 18-month-old mice. Representative photomicrographs of tyrosine hydroxylase staining in the ventral tegmental area of wild-type (A) and c-rel−/− (B) mice. (C) Densitometry analysis of tyrosine hydroxylase-positive cells. No significant decrease in tyrosine hydroxylase-positive neurons was evident in c-rel−/− mice. The data represent the mean ± SEM (n = 7 animals per group, *P < 0.05 versus wild-type mice). Representative photomicrographs are shown of ChAT staining in the medial septal area of wild-type (D) and c-rel−/− mice (E) and in the nucleus basalis magnocellularis of wild-type (G) and c-rel−/− (H) animals. Representative pictures of NeuN staining in the striatum of wild-type (L) and c-rel−/− (M) mice. No significant difference in the estimated number of ChAT-positive cells in the medial septal area (F) and the nucleus basalis magnocellularis (I) or NeuN-positive neurons in the striatum (N) was evident between the two groups. Data represent the means ± SEM (n = 6 animals per group, *P < 0.05 versus wild-type mice). Scale bars: in A = 350 µm for A and B; in D = 500 µm for D and E; in G = 350 µm for G and H; L = 1200 µm for L–M, l insert = 300 µm for l, m. MSA = medial septal area; NBM = nucleus basalis magnocellularis; STR = striatum; VTA = ventral tegmental area; wt = wild-type.
Figure 3
Figure 3
Molecular changes in the dorsal striatum. Low (A and B) and high magnification (C and D) of representative pictures of tyrosine hydroxylase immunostaining in caudate putamen sections from 18-month-old wild-type (A–C) and c-rel−/− mice (B–D). (E) Densitometric analysis revealed a significant decrease in the density of tyrosine hydroxylase-positive fibres in 18-month-old c-rel−/− mice. The data represent the mean ± SEM (n = 4 animals per group, *P < 0.05 versus wild-type mice). Scale bar in A = 500 µm for A and B; in C = 300 µm for C and D. (F) Representative immunoblotting of the dopamine transporter in the caudate putamen extracts of 18-month-old wild-type and c-rel−/− mice. Densitometry analysis showed a significant reduction in dopamine transporter levels in c-rel−/− when compared with wild-type animals. Results represent the mean ± SEM (n = 4 animals per group, *P < 0.05 versus wild-type mice). (G) Quantification of dopamine, dopamine metabolites (dihydroxylphenylacetic acid and homovanillic acid), NA, 5-hydroxytryptamine with relative metabolite (5-hydroxyindoleacetic acid) detected in caudate putamen extracts of 18-month-old mice by HPLC analysis. A significant decrease of dopamine and homovanillic acid levels was detected in c-rel−/− mice. Data are means ± SEM of three independent experiments (n = 5 animals for group. *P < 0.05 versus wild-type mice). Quantification of noradrenaline, dopamine with its metabolites (homovanillic acid and dihydroxylphenylacetic acid) and 5-hydroxytryptamine with its metabolite (5-hydroxyindoleacetic acid) detected in the caudate putamen extracts of 18-month-old mice by HPLC analysis. A significant decrease in dopamine and homovanillic acid levels was detected in c-rel−/− mice. Data represent the mean ± SEM of three independent experiments (n = 5 animals per group, *P < 0.05 versus wild-type mice). DA = dopamine; DAT = dopamine transporter; DOPAC = dihydroxylphenylacetic acid; HVA = homovanillic acid; 5HT = 5-hydroxytryptamine; 5HIAA = 5-hydroxyindoleacetic acid; NA = noradrenaline.
Figure 4
Figure 4
Accumulation of α-synuclein in 18-month-old c-rel−/− and wild-type mice. (A and D) Representative photomicrographs showing tyrosine hydroxylase (blue) and α-synuclein (brown) double staining in the substantia nigra of 18-month-old wild-type (A and C) and c-rel−/− mice (B and D). Note that the increase of brown α-synuclein staining in the c-rel−/− mice (d) completely covered the blue tyrosine hydroxylase immunoreactivity observed in wild-type mice (c). (E-L) Representative photomicrographs showing α-synuclein (red), thioflavin S (green) and tyrosine hydroxylase (blue) triple staining in the substatia nigra of c-rel−/− (E–H) and wild-type (I–L) mice. Note that the α-synuclein-positive aggregates in the brain of c-rel−/− mice were also thioflavin S immunopositive, as visualized in the high-magnification squares in panels E–H. Scale bars: in A = 3 mm for A and B; in C = 750 µm for C and D; in insert c = 10 µm for c and d; in E = 50 µm for E–L; in insert e = 10 µm for e–h. Panels A–L are representative of three independent experiments (n = 3 animals per group). (M) Representative western blot showing sequential α-synuclein extraction from diverse brain areas in 18-month-old wild-type and c-rel−/− (-/-) mice. Recombinant α-synuclein (+) solution was used as positive control. Note the increase in α-synuclein-immunopositive bands in the mesencephalon of c-rel−/− mice when compared with wild-type animals, as well as the α-synuclein immunopositive band in the urea extracts from c-rel−/− mice. (N) Densitometric analysis of α-synuclein-immunopositive bands in the TBS and TBS/Triton fractions. Note the significant increase of α-synuclein in the mesencephalon of c-rel−/− mice. Data represent the mean ± SEM (n = 3 animals per group, *P < 0.05 versus wild-type mice). (O) Representative western blot showing the increase in the α-synuclein immunoreactive bands in the urea + SDS fractions from the mesencephalon of c-rel−/− mice. Similar results were obtained in three separate experiments.
Figure 5
Figure 5
DMT1 immunoreactivity and iron staining in mesencephalon of 18-month-old c-rel−/− and wild-type mice. Tyrosine hydroxylase staining (A and B) and diaminobenzidine enhanced Perl iron staining (C–F) in sections of 18-month-old wild-type (A, C and E) and c-rel−/− mice (B, D and F). Higher magnifications of Perl iron staining are in panels E and F. While the number of tyrosine hydroxylase-positive neurons decreased, the iron staining significantly increased in the substantia nigra pars compacta and reticulata of c-rel−/− mice compared with wild-type mice. Representative immunoblotting of the ferrous iron transporter DMT1 in the striatal and mesencephalic extracts of 18-month-old wild-type and c-rel−/− mice (G). Densitometric analysis of DMT1 relative to GAPDH (H). DMT1 60 and 90 kDa bands significantly increased in the mesencephalon of c-rel−/− mice (-/-) compared with wild-type animals. Significant difference in the DMT1 90 kDa band was detected in the striatum. Values represent the mean ± SEM (n = 3 animals per group, *P<0.001 versus wild-type mice) (F). Scale bar in A = 600 µm for A–D; in E = 75 µm for E and F.
Figure 6
Figure 6
CD11b and GFAP immunoreactivity in 18-month-old c-rel−/− and wild-type mice. Pictures of the substantia nigra pars compacta (A and B) and striatum (C and D) sections from 18-month-old wild-type (A and C) and c-rel−/− mice (B and D) illustrating CD11b microglial immunostaining. (E) The quantification of CD11b is expressed as a percentage of grey values. There was significant microglial activation in both the striatum and the substantia nigra pars compacta of c-rel−/− mice. Images of the substantia nigra pars compacta (F and G) and striatum (H and I) sections from 18-month-old wild-type (F and H) and c-rel−/− mice (G and I) showing GFAP astroglial immunostaining. (L) The percentage of GFAP-positive cells. No evident changes in astroglial activation were found in c-rel−/− mice. Scale bars: in A = 200 µm for A–D; in C’ and F = 50 µm for A’–D’ and F–I. Data represent the mean ± SEM (n = 5 animals per group, *P < 0.05, **P < 0.005 versus wild-type mice). SNc = sunstantia nigra pars compacta.
Figure 7
Figure 7
Spontaneous motor and locomotor activity in c-rel−/− and wild-type mice. (A–C) Mice were tested at 4 months (A, n = 9–10), 12 months (B, n = 17–18) and 18 months (C, n = 14–18). The data represent the mean ± SEM of the total motor and locomotor activity counts registered during the 60-min observation (*P < 0.05 versus wild-type mice). (D) Mice at 18 months were tested by a video-tracking system in a open field arena for 1 h. c-rel−/− mice were less active as assessed by the total distance moved (cm) (n = 6 animals per group, *P < 0.05 versus wild-type mice). (E) The wild-type and c-rel−/− mice at 18 months were maintained in a PhenoTyper cage and continuously monitored for six consecutive days. The total distance moved (cm) in a day increased after 4 days of observation in the wild-type group but not in the c-rel−/− group (n = 11 animals per group, *P < 0.05 versus wild-type mice).
Figure 8
Figure 8
CatWalk gait analysis of c-rel−/− and wild-type mice. The data represent the mean ± SEM of three runs per animal (n = 11 animals per group). (A) Lower swing speed (m/s) and higher base of support for both the front (B) and hind (C) paws was observed in c-rel−/− mice (*P < 0.05 versus wild-type mice). Higher percentage of time spent to reach the maximum contact (D) of the front limbs was observed in c-rel−/− mice (**P < 0.001 versus wild-type mice). A lower print length of the front paws (E) was observed in c-rel−/− mice (*P < 0.05 versus wild-type mice).
Figure 9
Figure 9
l-DOPA treatment ameliorates the motor deficits in c-rel−/− mice (18 months). l-DOPA (20 mg/kg) and benserazide hydrochloride (12.5 mg/kg), or vehicle, were administered to mice 1 h before the beginning of the behavioural tests. (A) l-DOPA treatment increased the spontaneous locomotor activity evaluated as total distance moved (cm) in the open field during 1 h observation (*P < 0.05 versus vehicle). l-DOPA supplementation improved the gaiting performance by increasing the swing speed (m/s) (B), by reducing the base of support for both the front (C) and hind paws (D) and by reducing the percentage of time spent to reach the maximum contact (E) (*P < 0.05 versus vehicle). l-DOPA treatment increased, though not significantly, the fore paw print length (mm) (F). veh = vehicle. The data represent the mean±SEM of three runs per animal (n = 6 animals per group).
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